I. Field of the Invention
This invention relates to the field of immunoassays, more particularly to reagents and methods useful for the rapid and sensitive quantification of the peptide hormone hBNP in a biological fluid such as plasma or serum.
II. Description of the Prior Art
BNP is a cardiac derived peptide hormone that circulates in the blood and exerts potent cardiovascular and renal actions. BNP is structurally similar to two other cardiac peptides, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). Porcine BNP was isolated from pig brain (Sudoh et al., Nature, 332:78-81, 1988) and, hence, was given the name "brain natriuretic peptide". In man the cardiac ventricle is the primary site of BNP synthesis. The sequence of human BNP (hBNP) was originally determined by the isolation and characterization of DNA clones from human genomic libraries (U.S. Pat. No. 5,114,923). hHBNP is synthesized, in vivo, as a 108 amino acid precursor that is enzymatically cleaved to yield the mature hBNP peptide. Mature hBNP consists of a 32 amino acid peptide containing a 17 amino acid ring structure formed by two disulfide bonds (see FIG. 1).
Elevated expression of ventricular hBNP mRNA has been reported in congestive heart failure patients as compared to normal controls. Consistent with the increase in ventricular mass and expression of BNP mRNA, hBNP levels are elevated in patients with congestive heart failure and appear to correlate with disease severity. Plasma hBNP is also believed to provide a valuable predictive marker for heart disease. Elevated plasma hBNP has been reported in heart disease, following acute myocardial infarction and during symptomless or subclinical ventricular dysfunction (Mukoyama et al., J. Clin. Invest., 87:11402-1412, 1991) (Motwani et al., Lancet, 341:1109-1113, 1993) (Yoshibayashi et al., New Eng. J. Med., 327:434, 1992). Reports from two major therapeutic trials, the SAVE trial (New Eng. J. Med., 327:669-677, 1992) and the SOLVD trial (New Eng. J. Med., 327:685-691, 1992) suggested that diagnosis and appropriate treatment of patients with asymptomatic left ventricular dysfunction could significantly reduce the incidence of fatal and non-fatal cardiovascular events and related hospitalizations.
For use in a clinical laboratory setting, it would be highly desirable to provide a diagnostic assay for hBNP which is sufficiently sensitive to measure clinically relevant titers of hBNP, sufficiently simple that it can be automated, requires a minimum amount of time to complete and preferably does not require the use of reagents having limited shelf lives. Normal levels of hBNP in plasma are quite low, on the order of 1 to 20 pg/mL. Typically, radioimmunoassays are used to measure titers in this range, although the use of radioactive reagents requires special handling, adds steps to the assay and involves the use of material having limited shelf life. A radioimmunoassay is commercially available for measuring hBNP, however, in addition to requiring the use of radioactive reagents, it is complex and cumbersome, requiring an extraction step, precipitation and several centrifugation steps, and takes several days to complete. European Patent Application No. 0 542 255 describes a radioimmunoassay for hBNP.